title: Purification, characterization and kinetic studies of a novel poly(β) hydroxybutyrate (PHB) depolymerase PhaZ(Pen) from Penicillium citrinum S2.
abstract: A fungal isolate, identified as Penicillium citrinum S2, produced ≈1 U/mL of PHB depolymerase by 72 h when grown in BHM containing 0.2%, w/v PHB, pH 6.0 at 30 °C. Partial purification of an extracellular poly(-β-)hydroxybutyrate (PHB) depolymerase PhaZ( Pen ) from P. citrinum S2 by two steps using ammonium sulphate (80% saturation) and affinity chromatography using concanavalin A yielded 16.18-fold purity and 21.53% recovery of protein. The enzyme was composed of three polypeptide chains of 66, 43 and 20 kDa, respectively, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. All the three bands stained positive for glycoprotein by PAS staining. Optimum enzyme activity was detected at pH 6.0 and 50 °C. The enzyme was stable between pH 4.0 and 7.0 at 50 °C, 2 h. β-hydroxybutyrate monomer was detected as the major end product of PHB hydrolysis. The enzyme also showed distinct behaviour towards different inhibitors tested, which suggests the role of serine, serine residue, carboxyl group, tyrosine and sulfhydryl groups in its active site.
authors.affiliation: Department of Microbiology, Centre for Post Graduate Studies, Jain University, 18/3, 9th Main, Jayanagar 3rd Block, Bangalore 560011, India. sk2410@yahoo.co.uk
authors.forename: Srividya
authors.initials: S
authors.lastname: Shivakumar
authors.forename: Sneha Jani
authors.initials: SJ
authors.lastname: Jagadish
authors.forename: Hardik
authors.initials: H
authors.lastname: Zatakia
authors.forename: Jayasmita
authors.initials: J
authors.lastname: Dutta
mesh_terms: D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D020134:Catalytic Domain; D002846:Chromatography, Affinity; D003208:Concanavalin A / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D010407:Penicillium / Q000201:enzymology*; D012694:Serine; D013379:Substrate Specificity; D013696:Temperature; D014443:Tyrosine
chemical_list: D008670:Metals; D003208:Concanavalin A; D014443:Tyrosine; D012694:Serine; D002265:Carboxylic Ester Hydrolases; C022417:poly-beta-hydroxybutyrate depolymerase